Microbial transformation of cadina-4,10(15)-dien-3-one, aromadendr-1(10)-en-9-one and methyl ursolate by Mucor plumbeus ATCC 4740

Article abstract of DOI:10.1016/S0031-9422(01)00486-1

The sesquiterpenes cadina-4,10(15)-dien-3-one (1) and aromadendr-1(10)-en-9-one (squamulosone) (14) along with the triterpenoid methyl ursolate (21) were incubated with the fungus Mucor plumbeus ATCC 4740. Substrates 1,14 and ursolic acid (20) were isolated from the plant Hyptis verticillata in large quantities. M. plumbeus hydroxylated 1 at C-12 and C-14. When the iron content of the medium was reduced, however, hydroxylation at these positions was also accompanied by epoxidation of the exocyclic double bond. In total nine new oxygenated cadinanes have been obtained. Sesquiterpene 14 was converted to the novel 2α,13-dihydroxy derivative along with four other metabolites. Methyl ursolate (21) was transformed to a new compound, methyl 3β,7β,21β-trihydroxyursa-9(11),12-dien-28-oate (22). Two other triterpenoids, 3β,28-dihydroxyurs-12-ene (uvaol) (23) and 3β,28-bis(dimethylcarbamoxy)urs-12-ene (24) were not transformed by the microorganism, however.

Full text of DOI:10.1016/S0031-9422(01)00486-1

Phytochemistry 59 (2002) 479–488
www.elsevier.com/locate/phytochem
Microbial transformation of cadina-4,10(15)-dien-3-one,
aromadendr-1(10)-en-9-one and methyl ursolate by
Mucor plumbeus ATCC 4740
Dwight O. Collinsa, Peter L.D. Ruddocka, Jessica Chiverton de Grassea,
William F. Reynoldsb, Paul B. Reesea,*
aDepartment of Chemistry, University of the West Indies, Mona, Kingston 7, Jamaica, West Indies
bDepartment of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6
Received 4 June 2001; received in revised form 13 October 2001
Dedicated to Professor Sir John Cornforth, University of Sussex, as he enters his 85th year
Abstract
The sesquiterpenes cadina-4,10(15)-dien-3-one (1) and aromadendr-1(10)-en-9-one (squamulosone) (14) along with the triterpe-
noid methyl ursolate (21) were incubated with the fungus Mucor plumbeus ATCC 4740. Substrates 1, 14 and ursolic acid (20) were
isolated from the plant Hyptis verticillata in large quantities. M. plumbeus hydroxylated 1 at C-12 and C-14. When the iron content
of the medium was reduced, however, hydroxylation at these positions was also accompanied by epoxidation of the exocyclic
double bond. In total nine new oxygenated cadinanes have been obtained. Sesquiterpene 14 was converted to the novel 2a,13-
dihydroxy derivative along with four other metabolites. Methyl ursolate (21) was transformed to a new compound, methyl
3b,7b,21b-trihydroxyursa-9(11),12-dien-28-oate (22). Two other triterpenoids, 3b,28-dihydroxyurs-12-ene (uvaol) (23) and 3b,28-
bis(dimethylcarbamoxy)urs-12-ene (24) were not transformed by the micro-organism, however. # 2002 Elsevier Science Ltd. All
rights reserved.
Keywords: Mucor plumbeus ATCC 4740; Cadinane; Aromadendrane; Ursane; Biotransformation; Hydroxylation; Hyptis verticillata; Sesquiterpene;
Triterpene
1. Introduction
formicarius elegantulus than the naturally occurring
compound. The fungal metabolism of 14 has also been
investigated using Curvularia lunata ATCC 12017, pro-
ducing seven new aromadendranes (Collins et al., 2001).
In our ongoing programme to examine the metabolism
of plant derived terpenes and their derivatives by fungi
(Hanson et al., 1994; Buchanan and Reese, 2000), com-
pounds 1, 14 and methyl ursolate (21) were incubated
with Mucor plumbeus (formerly M. spinosus) ATCC
4740. Additionally, the incubation of substrate 1 in two
media, a low iron (LIM) and a high iron medium (HIM)
(Aranda et al., 1991a), highlighted the effect of varying
the iron content of the medium upon the outcome of
bioconversion. Seven metabolites emerged with the use
of HIM and nine with LIM. Four of the compounds
were common to both fermentations. Interestingly
epoxidation of the exocyclic double bond only occurred
with the use of LIM. Transformations of substrates 14
and 21 were performed only in HIM. Five congeners
were obtained when 14 was fed to the fungus, only one
Hyptis verticillata Jacq. (Labiatae) has been used tra-
ditionally in the treatment of eczema, psoriasis, scabies,
athlete’s foot, rheumatoid arthritis and a host of cold-
related problems (Morton, 1981; Ayensu, 1981; Stanley,
1928). A number of compounds have been isolated and
characterised from the plant (German, 1971; Novelo et
al., 1993; Kuhnt et al., 1994, 1995), including the terpenes
cadina-4,10(15)-dien-3-one (1) (Porter et al., 1995), aro-
madendr-1(10)-en-9-one (squamulosone) (14) (Collins et
al., 2001) and ursolic acid (20) (Porter et al., 1995). A
previous incubation of 1 with the fungus Beauveria
bassiana ATCC 7159 resulted in the isolation of nine
metabolites (Buchanan and Reese, 2000), three of which
were more potent against the sweet potato weevil Cylas
* Corresponding author. Tel.: +1-876-927-1910; fax: +1-876-977-
1835.
E-mail address: pbreese@uwimona.edu.jm (P.B. Reese).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00486-1

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D.O. Collins et al. / Phytochemistry 59 (2002) 479–488
of which was new. Bioconversion of the triterpenoid
methyl ursolate (21) gave a novel compound. Attempts
at the transformation of two other triterpenoids: 3b,28-
dihydroxyurs-12-ene (uvaol) (23) and 3b,28-bis(di-
methylcarbamoxy)urs-12-ene (24) were unsuccessful.
M. plumbeus has been employed in the metabolism of
a number of terpene substrates (Aranda et al., 1991a,b,
1992; Azerad, 2000; Boaventura et al., 1995; Guillermo
et al., 1997; Fraga et al., 1998, 2001; Arantes et al.,
1999a,b,c,d; Arantes and Hanson, 1999; Maurs et al.,
1999). This list, while not extensive, includes the trans-
formation of three aromadendrane derivatives (Guil-
lermo et al., 1997). Fungal transformations of triterpenes
are extremely rare and none have been reported on
ursane substrates or by M. plumbeus.
cadin-10(15)-ene (1) and (4S)-3b,12-dihydroxycadin-10
(15)-ene (8). Compounds 3, 4 and 5 were identified by
comparison of their spectral data with those in the
literature. Previously, however, metabolite 4 had been
mistakenly identified as the 13-hydroxy derivative
(Buchanan and Reese, 2000). This correction was based
on the accepted principle that hydroxyl insertion at C-1
will cause upfield shifts of up to 6 ppm for C-3 (Silver-
stein and Webster, 1998), and as such the chemical
shifts of 4 are consistent with hydroxylation at C-14 and
not C-13. The assignments were aided by the fact that
the signals for the C-13 and-14 methyls were distinct,
having a difference of ꢀ6 ppm. A repeat fermentation
of 1, this time using LIM, yielded nine cadinanes only
four of which (congeners 4, 5, 6 and 7) were obtained in
common with the initial fermentation. The new meta-
bolites were identified as 13-hydroxycadina-4,10(15)-
dien-3-one (9), (4S)-10a,15-epoxy-12-hydroxycadinan-3-
one (10), (4S)-10a,15-epoxy-14-hydroxycadinan-3-one
(11), (4S)-10a,15-epoxy-3a,14-dihydroxycadinane (21)
and (4S)-3a,10a,12,15-tetrahydroxycadinane (13).
The IR absorption band at 3429 cmꢁ1 for 1 revealed
the presence of a hydroxyl group. The new functionality
was placed at C-14 based on a comparison of the che-
mical shifts of 2 with those of 1.
2. Results and discussion
The incubation of 1 with M. plumbeus in HIM produced
seven metabolites that were identified as 14-hydroxy-
cadina-4,10(15)-dien-3-one (2), (4S)-12-hydroxycadin-
10(15)-en-3-one (3), (4S)-14-hydroxycadin-10(15)-en-3-
one (1), (4S)-3a-hydroxycadin-10(15)-ene (2), (4S)-3a,12-
dihydroxycadin-10(15)-ene (2), (4S)-3a,14-dihydroxy-

D.O. Collins et al. / Phytochemistry 59 (2002) 479–488
481
The reduction of the a,b-unsaturated system in 6 to
form the saturated alcohol was proposed by the absence
of the olefinic resonances from its 13C NMR spectrum.
The latter contained two resonances corresponding to
hydroxyl bearing carbons, one of which was assigned to
C-3. The stereochemistry of this hydroxyl at C-3 was
determined to be a based on the small coupling constant
of H-3 (J=2.9 Hz). The (S)-configuration of the C-11
methyl followed from its relatively large resonance value
(Buchanan and Reese, 2000). The other hydroxyl,
attached to a tertiary carbon, was assigned to C-12.The
chemical shifts of the geminal dimethyls are consistent
with this placement.
Congener 7, having a molecular formula of C15H26O2
([M]+=238.1939) as suggested by HR(EI)MS data,
differed from 6 in that the reduction of the enone to the

482
D.O. Collins et al. / Phytochemistry 59 (2002) 479–488
3a-alcohol was accompanied by C-14 hydroxylation.
Compound 8 was found only to be the C-3 epimer of 6.
In compound 1 the substrate had undergone a previously
unreported hydroxylation at C-13 of the isopropyl
group.
stereochemistry was again suggested by the low value of
the H-3 coupling constant (J=2.5 Hz), while the adja-
cent C-11 methyl possessed an (S)-configuration as in
1
the case of 6. HMQC, HMBC and H–1H COSY data
were used to confirm the proposed structure. The
stereochemistry of the epoxide, determined from T-
ROESY data, was the same as that in 10.
Data from the HR(EI)MS of 10 suggested a mole-
cular formula of C15H24O3 ([M]+=252.1726). The
increase in the chemical shift of the carbonyl to 211.9 ppm
as compared with that of 1 indicated a loss of conjugation.
Notable was the absence of the olefinic carbons repre-
senting an exocyclic double bond and the appearance of a
sharp band at 1186 cmꢁ1 in the IR spectrum which corre-
sponded to an epoxide function. The presence of the
epoxy moiety was suggested by two new C–O resonances
(49.3 and 60.5 ppm) in the 13C NMR spectrum. An IR
band at 3462 cmꢁ1 suggested that the molecule also con-
tained a hydroxy function. This tertiary hydroxyl (ꢀC 73.9)
was accommodated by C-12. The stereochemical assign-
ments of the epoxide as well as the C-11 methyl were aided
significantly by T-ROESY data. For example the
enhancements of Hax-2 (ꢀ 1.84) or Hax-9 (ꢀ 1.42) when
either HS-15 (ꢀ 2.80) or HR-15 (ꢀ 2.48) was irradiated
firmly fixed the stereochemistry of the epoxide as a.
Compound 11, when analysed by HR(EI)MS, also
furnished an m/z of 252.1726 ([M]+) that translated into a
molecular formula of C15H24O3. In this case, however, the
1H NMR spectrum revealed the presence of only two
methyl groups within the structure. Like 10, three different
functionalities were apparent: a carbonyl (1713 cmꢁ1), an
epoxide (1230 cmꢁ1), and a hydroxyl (3421 cmꢁ1). This
hydroxyl group (ꢀC 66.5) was found to reside on C-14.
Metabolite 12 had comparable chemical shifts to 11
for ring B, but the absence of a carbonyl in ring A was
quite evident from its 13C NMR and IR spectra. With a
molecular formula of C15H26O3 ([M]+=254.1882), a
hydroxyl function indubitably existed at C-3. Its a-
Absent from the IR spectrum of congener 13 were
bands characteristic of carbonyl or epoxide functions.
This information along with that gleaned from the 13C
NMR data indicated that 13 contained four hydroxyl
1
groups. HMQC, HMBC and H–1H COSY data were
quite useful in establishing the connectivities within the
molecule as the chemical shifts varied considerably from
other metabolites. The stereochemistry of the groups
was again established by T-ROESY data. Metabolite 13
is perceived to have transited through an initial 10,15-
epoxide that opened to form the vicinal diol.
The incubation of aromadendr-1(10)-en-9-one (squa-
mulosone) (14) with M. plumbeus yielded five metabo-
lites that were identified as 2b-hydroxyaromadendr-
1(10)-en-9-one (15), 2a-hydroxyaromadendr-1(10)-en-9-
one (16), 13-hydroxyaromadendr-1(10)-en-9-one (17),
2b,13-dihydroxyaromadendr-1(10)-en-9-one (18), and
2a,13-dihydroxyaromadendr-1(10)-en-9-one (19). Com-
pounds 15–18 were all identified by comparison of their
spectral data with those of known metabolites (Collins
et al., 2001). HR(EI)MS data of 19 suggested a mole-
cular formula of C15H22O3 ([M]+=250.1569) and this
indicated the presence of two additional hydroxyl
groups. The two new oxygen-bearing carbons observed
in the 13C NMR were present as a methine (ꢀC 74.2) and
a methylene (ꢀC 71.9) and were assigned to C-2 and C-
13, respectively. The stereochemistry of the new C-2
function followed from the large coupling constant
(14.6 Hz) for H-2 (Collins et al., 2001).
Table 1
Carbon-13 NMR resonances for cadina-4,10(15)-dien-3-one (1), squamulosone (14) and metabolites of both
Compounds
1
2
6
7
8
9
10
43.6
11
41.9
12
13
14
19
C-1
C-2
45.31
41.4
45.1
41.3
46.2
36.6
70.1
45.0
36.5
70.4
45.9
38.3
75.9
45.21a
41.2
199.9
37.1
32.5
69.6
39.9
34.9
71.6
166.4
34.1
32.2
163.7
74.7
42.3
40.4
211.9
40.2
212.0
C-3
C-4
199.9
135.5
148.4
45.29
199.9
135.735.9
146.0
44.6
35.9
40.0
135.4
44.5
44.2
35.8
38.4
.2 37 32.4
33.745.2
C-5
C-6
34.3
38.5
31.9
39.1
39.9
45.0
146.3
45.18a
42.1
46.4
39.8
46.0
31.9
42.6
44.2
19.6
41.5
31.3
28.0
C-745.1
C-8
C-9
40.1
53.1
42.8
53.0
45.0
52.4
42.2
42.3
39.3
22.8
26.3
35.5
26.6
35.2
31.0
36.4
26.8
36.4
31.0
36.3
26.2
35.3
27.9
34.5
60.6
14.4
73.9
24.1
32.4
49.3
23.4
34.4
23.7
35.0
16.9
30.6
41.9
201.0
41.5
201.2
C-10
C-11
C-12
C-13
C-14
C-15
149.8
16.0
26.4
149.6
15.9
34.6
152.6
18.6
74.2
24.2
31.7
103.6
152.8
18.6
34.6
10.2
67.4
103.8
151.7149.7 60.5
61.2 5.2
14.3
34.9
7
130.3
133.9
18.5
74.2
15.9
38.6
18.5
34.4
10.2
67.0
49.9
18.1
76.1
28.33a
28.29a
66.1
25.731.9
16.0
28.2
11.7
71.9
14.4
16.4
15.2
21.5
10.2
66.4
24.2
32.0
68.0
15.1
10.2
66.5
14.9
15.4
105.3
105.5
104.2
105.2
49.3
a
Assignments are interchangeable within a column.

D.O. Collins et al. / Phytochemistry 59 (2002) 479–488
483
Three triterpenoids: methyl ursolate (21), 3b,28-di-
Table 2
Carbon resonances for ursane triterpenoids
hydroxyurs-12-ene (uvaol) (23) and 3b,28-bis(dimethyl-
carbamoxy)urs-12-ene (24) were also incubated with
M. plumbeus. While substrates 23 and 24 were recovered
untransformed from the fermentation broth, 21 under-
went hydroxylation to give methyl 3b,7b,21b-trihydroxy-
ursa-9(11),12-dien-28-oate (22) as the sole product.
Pivotal in the structural elucidation of 22 was the com-
21
38.5
22
37.5
23
38.7
24
38.4
C-1
C-2
27.0
78.7
38.5
55.1
18.1
27.8
78.5
26.1
81.8
27.2
79.0
C-3
C-4
38.738.0
47.9
29.3
38.0
C-5
C-6
55.2
18.2
55.1
18.3
1
plete resolution of all methyl groups in the H NMR
1.632.8C-7323.82.7
C-8
C-9
7
spectrum, a rarity for triterpenes. As such HMBC cor-
relations were used to unambiguously assign the reso-
nances. The presence of two additional hydroxyl groups
and a second double bond was substantiated by a sug-
gested molecular formula of C31H48O5 ([M]+=500.3502).
Again T-ROESY data aided stereochemical proposi-
tions and indicated that ring E was slightly distorted
from its normal chair conformation. The two new
hydroxyls were equatorial. The formation of 21 is
thought to have proceeded through an 11-hydroxy
intermediate that readily eliminated to form the con-
jugated diene. It is not sure, however, if this reaction
occurred at an early or late stage in the metabolism of
the substrate. The failure of the fungus to effect reaction
on either substrate 23 or 24 may be a direct result of
polarity of the former and size of the latter.
In summary sesquiterpenes 1 and 14 and triterpenoids
21, 23 and 24 were incubated with the fungus M. plum-
beus. Substrate 1 yielded seven metabolites in a high
iron medium (HIM) and nine metabolites in a low iron
medium (LIM). Epoxidation of the 10(15)-exocyclic
double bond occurred exclusively in the latter. Nine new
cadinanes were thus produced. Substrate 14 was trans-
formed to five metabolites, only one of which was new.
Here reactions were centred on C-2 and C-13 with little
stereoselectivity. Of the three triterpenoids, only one,
21, was transformed by the fungus and this resulted in
the isolation of a single, but novel metabolite.
39.3
47.4
41.8
154.9
40.0
47.5
40.0
47.6
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-2723.4
C-28
C-29
C-30
OCH3
36.739.1
23.1
36.7 36.8
a
116.723.5
123.9
139.0
48.8
23.33a
125.0
138.7
42.0
125.4
137.9
41.8
125.4
138.1
41.9
24.1a
23.4
37.2
27.8
24.0
30.2
25.8
26.0
23.27a
36.8
47.9
52.751.0
38.8
48.1
54.4
54.0
39.3
37.6
39.4
39.4
38.746.6
30.5
36.4
39.2
71.1
44.5
30.6
35.8
28.1
17.3
30.6
35.1
28.1
15.7
28.0
15.5
28.2
15.6
15.3
16.713.8
23.3
24.9
15.715.6
16.7 16.7
23.2
18.3
177.9
16.9
21.0
176.5
17.1
15.8
51.9
72.1
16.9
21.3
69.9
17.3
21.3
51.3
OCO-N(CH3)2-28
OCO-N(CH3)2-3
OCO-N(CH3)2-28
OCO-N(CH3)2-3
35.8
36.3
156.7
156.9
a
Assignments are interchangeable within a column.
on a VG 70-250S or a Kratos MS50 instrument at an
ionising voltage of 70 eV. Column chromatography was
performed with Kieselgel silica (40–63 mm). Compounds
on TLC plates were visualised by the use of the ammo-
nium molybdate/sulfuric acid spray reagent followed by
heating at 120 ꢂC. Mucor plumbeus ATCC 4740 was
obtained from the American Type Culture Collection,
Rockville, MD, USA. Petrol refers to the petroleum
3. Experimental
1H and 13C NMR spectra were generated in deuterated
chloroform at 200 and 50 MHz respectively using a Bru-
ker AC200 instrument. 2D NMR data were generated on
a Varian Unity 500 spectrometer. Tetramethylsilane
(TMS) was used as the internal standard. 13C NMR
assignments for the cadinanes and aromadendranes are
listed in Table 1 while those for the ursanes are reported
in Table 2. Melting points were measured on a Thomas–
Hoover capillary melting point apparatus. Infrared data
were obtained on a Perkin Elmer FTIR Paragon 1000
instrument using KBr disks. The UV spectra were
determined on a Hewlett Packard HP 8452A spectro-
photometer. Optical rotations were acquired on a Per-
kin Elmer 241 polarimeter. High-resolution electron
impact mass spectrometry [HR(EI)MS] was performed
ꢂ
fraction boiling between 60 and 80 C.
3.1. Generation of substrates
Hyptis verticillata plant material was collected in St
Andrew, Jamaica. A voucher specimen was deposited in
the Botany Herbarium, UWI (accession number 33483).
Two separate collections of the plant material were
necessary to isolate the different compounds. In the
first, the green leaves and stems (17.61 kg) were
collected in the first quarter of the year, chopped and
extracted twice with CH2Cl2 (47l) at room temperature.

484
D.O. Collins et al. / Phytochemistry 59 (2002) 479–488
The extracts were pooled and concentrated in vacuo to
produce a dark green gum (145.5 g). The crude extract
was chromatographed on silica gel using 3% EtOAc in
petrol to give eight major fractions (A–H). The moder-
ately nonpolar fraction B (42.3 g) was subjected to further
purification by column chromatography using 0.5%
EtOAc in petrol. This led to the isolation of a cadina-
4,10(15)-dien-3-one (1) (4.7g).
gen through the solution. The solution was con-
centrated in vacuo, chromatographed using 10% ethyl
acetate in petrol, and the product was recrystallised
from acetone to yield methyl 3b-hydroxyurs-12-en-28-
oate (methyl ursolate) (21) (2.14 g, 4.6 mmol).
3.2.1. Methyl 3ꢃ-hydroxyurs-12-en-28-oate (21)
Amorphous crystals from acetone, mp 166–168 ꢂC;
25
31
ꢂ
½ꢁŠD : +61ꢂ (c=19.8, CHCl3) [lit. mp 168–170 C, ½ꢁŠD :
+56.0ꢂ (Gupta and Mahadevan, 1968); IR ꢂmax cmꢁ1
3453, 1726, 1459; H NMR: ꢀ 0.74 (3H, s, H-26), 0.78
1
3.1.1. Cadina-4,10(15)-dien-3-one (1)
Needles from acetone, mp 74–75 ꢂC; ½ꢁŠD25: +134ꢂ
(3H, s, H-24), 0.86 (3H, d, J=6.2 Hz, H-29), 0.92 (3H,
s, H-25), 0.93 (3H, d, J=6.6 Hz, H-30), 0.98 (3H, s, H-
23), 1.08 (3H, s, H-27), 3.20 (1H, t, J=6 Hz, H-3), 3.60
(3H, s, CO2Me), 5.24 (1H, t, J=3.7Hz, H-12).
(c=10.3, CHCl3) [lit. mp 79–80 ꢂC, ½ꢁŠD25: +127ꢂ
(c=1.6, CHCl3) (Porter et al., 1995)]; IR ꢂmax cmꢁ1
1
2950, 1680, 890; H NMR: ꢀ 0.80 (3H, d, J=12 Hz, H-
14), 1.00 (3H, d, J=12 Hz, H-13), 1.80 (3H, m, w=2
=17Hz, H-11), 2.25 (1H, m, w=2=38 Hz, H-12), 4.47
(1H, s, H-15), 4.73 (1H, s, H-15), 6.82 (1H, s, H-5).
Further elution in 1% EtOAc in petrol led to the iso-
lation of aromadendr-1(10)-en-9-one (14) (10.3 g).
3.2.2. 3ꢃ,28-Dihydroxyurs-12-ene (uvaol) (23)
Methyl 3b-hydroxyurs-12-en-28-oate (21) (1.4 g, 3.0
mmol) was dissolved in THF (40 ml) and lithium alumi-
nium hydride (0.5 g, 13.5 mmol) was added. The reaction
was refluxed for 3 h. The addition of water to the reaction
mixture was followed by extraction with EtOAc (3ꢃ50
ml) and concentration in vacuo to give 3b,28-dihydroxy-
3.1.2. Aromadendr-1(10)-en-9-one (14)
Needles from acetone, mp 50–51 ꢂC; ½ꢁŠD25: ꢁ202ꢂ
(c=1.43, CHCl3) [lit. mp 45–46 ꢂC, [ꢁ]D: ꢁ234ꢂ (c=1.2,
CHCl3) (Batey et al., 1971)]; IR ꢂmax cmꢁ1 2954, 2871,
1639 (C¼O), 1468, 1315; UV (MeOH) lmax (log ") 254
urs-12-ene (23) (1.25 g, 2.8 mmol): Needles from acetone,
25
ꢂ
ꢂ
mp 223–224 C; ½ꢁŠD : +67 .0 (c=8.1, CHCl3) [lit. mp
226–227 ꢂC, [ꢁ]D: +7 0 (c=2.6, CHCl3) (Orzalesi et al.,
1969)]; IR ꢂmax cmꢁ1 3358, 2927, 1457, 1043; H NMR:
ꢂ
1
1
(3.81); H NMR: ꢀ 0.71 (1H, ddd, J=5.2, 9.5, 11.8 Hz,
H-7), 0.82 (1H, dd, J=9.5, 11.1 Hz, H-6), 1.04 (3H, d,
J=6.5 Hz, H-15), 1.07(3H, s, H-13), 1.18 (3H, s, H-12),
1.42 (1H, m, w=2=26.7Hz, H-3), 1.78 (3H, bs, H-14),
ꢀ 0.79 (3H, s, H-24), 0.81 (3H, d, J=6.0 Hz, H-29), 0.93
(3H, d, J=3.8 Hz, H-30), 0.94 (3H, s, H-25), 0.99 (6H,
s, H-23, H-26), 1.10 (3H, s, H-27), 3.18 (1H, d, J=11.1
Hz, H-28), 3.20 (1H, m, w=2=12.0 Hz, H-3), 5.13 (1H,
t, J=3.8 Hz, H-12).
1.87(1H,
m, w=2=18.7Hz, H-3), 2.21 (1H,
m,
w=2=24.0 Hz, H-4), 2.34 (1H, m, w=2=22.3 Hz, H-2),
2.35 (1H, dd, J=11.8, 14.8 Hz, H-8), 2.62 (1H, bt,
J=11.0 Hz, H-5), 2.67(1H, bdd, J=8.2, 18.0 Hz, H-2),
2.81 (1H, dd, J=5.2, 14.8 Hz, H-8).
3.2.3. 3ꢃ,28-Bis(dimethylcarbamoxy)urs-12-ene (24)
3b,28-Dihydroxyurs-12-ene (23) (1.0 g, 2.3 mmol) was
dissolved in pyridine (2.3 ml, 2.3 g, 29.1 mmol) and dime-
thylcarbamyl chloride (19 ml, 16.3 g, 151.3 mmol) was
added. The reaction mixture was refluxed for 24 h, cooled
and then CuSO4 (aq) was added. The mixture was extrac-
ted with EtOAc (3ꢃ50 ml) and concentrated in vacuo to
yield 3b,28-bis(dimethylcarbamoxy)urs-12-ene (24) (900
mg, 2 mmol). Plates from acetone, mp 175–177 ꢂC; ½ꢁŠD25:
+61.9 (c=8.4, CHCl3); IR ꢂmax cmꢁ1 2927, 2870, 1704,
1492, 1397, 1190; EIMS m/z 584.4553 (1) [M]+, 569.4444
(1), 495.40494 (100), 406.3600 (8), 217.1916 (11), 216.1878
The green leaves and stems (12.1 kg) of the second
collection, done in the second quarter, were used to pro-
duce the concentrated dichloromethane (97g from 44.5 l).
ꢂ
The plant residue was dried at 40 C for 4 days, milled,
and then percolated with acetone (19.5 l). Removal of the
solvent gave a dark green gum (33.7g). The dichloro-
methane extract contained only very small quantities of
sesquiterpenes 1 and 14. The acetone extract was chro-
matographed on silica gel using progressively increasing
proportions of acetone in petrol to give five main frac-
tions (I–M). Fraction K (11.6 g) was subjected to fur-
ther purification by column chromatography using 60%
EtOAc in petrol. Ursolic acid (20) (5.6 g), thus isolated,
was characterised as its methyl ester (21).
1
(40.27), 204.1878 (13), 203.1800 (24); H NMR: ꢀ 0.81
(3H, d, J=4.7Hz, H-29), 0.87(3H, s, H-24), 0.91 (3H, s,
H-25), 0.94 (3H, d, J=6.6 Hz, H-30), 0.96 (3H, s, H-26)*,
1.01 (3H, s, H-23)*, 1.10 (3H, s, H-27), 2.90 (12H, bs, (2 x
(CH3)2N), 4.34 (1H, dd, J=5.1, 11.1 Hz, H-3), 5.13 (1H, t,
J=3.2 Hz, H-12). *Assignments may be interchanged.
3.2. Methyl 3ꢃ-hydroxyurs-12-en-28-oate (21)
To a suspension of crude ursolic acid (20) (2.18 g, 4.8
mmol) in CH2Cl2 (30 ml) was added excess ethereal
diazomethane. When the reaction was complete (TLC)
the excess diazomethane was removed by passing nitro-
3.3. Feeding protocol
The fungus was maintained on PDA slants. The high
iron (HIM) liquid growth medium contained per litre:

D.O. Collins et al. / Phytochemistry 59 (2002) 479–488
485
glucose (30 g), corn steep solids (5 g), sodium nitrate (2
3.4.4. (4S)-3ꢁ-Hydroxycadin-10(15)-ene (5) (HIM,
48.6 mg; LIM, 15.9 mg)
g), potassium chloride (0.5 g), magnesium sulfate (0.5 g)
and iron(II) sulfate (0.02 g) (Aranda et al., 1991a). The
only difference with the low iron (LIM) medium was a
change in the concentration of iron sulfate (0.002 g/l).
One 14 day old slant was used to inoculate three 500 ml
conical flasks each containing 125 ml of liquid medium.
The flasks were incubated at 200 rpm at 27 ꢂC. The
substrates 1 (1 g), 14 (1 g) and 21 (0.5 g), were each
dissolved in ethyl acetate (3 ml) and fed to the fungus 72
h after inoculation. The fermentation was allowed to
proceed for 10 days. The pH was measured and the
mycelium was filtered from the broth. The mycelium
was filtered and the broth was extracted with EtOAc
(3ꢃ500 ml). The mycelium was homogenised in EtOAc.
The extracts were dried with sodium sulfate,
concentrated in vacuo, and analysed by thin layer
chromatography.
Cubes from acetone, mp 132–134 ꢂC; ½ꢁŠD25: +14.7ꢂ
(c=1.9, CHCl3) which was identified by comparison of
its physical and spectral data with those in the literature
(Buchanan and Reese, 2000).
3.4.5. (4S)-3ꢁ,12-Dihydroxycadin-10(15)-ene (6)
(HIM, 88.6 mg; LIM, 41.8 mg)
Cubes from acetone, mp 91–92 ꢂC; ½ꢁŠD25:ꢁ0.2ꢂ
(c=5.2, CHCl3); IR ꢂmax cmꢁ1 3385, 2924, 1710; EIMS
m/z (rel. int.) 238.1932 (1) [M]+, 220.1828 (26),
202.1728 (29), 180.1517 (16), 162.1413 (76), 159.1178
(40), 147.1177 (66), 133.1019 (32); 1H NMR: ꢀ 0.97(3H,
d, J=7.0 Hz, H-11), 1.15 (3H, s, H-14), 1.21 (3H, s, H-
13), 3.90 (1H, d, J=2.9 Hz, H-3), 4.51 (1H, bs, H-15),
4.64 (1H, d, J=1.3 Hz, H-15).
3.4.6. (4S)-3ꢁ,14-Dihydroxycadin-10(15)-ene (7)
(HIM, 70.8 mg; LIM, 44.5 mg)
3.4. Incubation of cadina-4,10(15)-dien-3-one (2)
Cubes from acetone, mp 99–101 ꢂC; ½ꢁŠD25: +0.6ꢂ
(c=1.68, CHCl3); IR ꢂmax cmꢁ1 3410, 2924, 1646, 1450
cmꢁ1; EIMS m/z (rel. int.) 238.1939 (1) [M]+, 220.1829
(32), 189.1646 (40), 162.1066 (73), 161.1333 (100),
Cadina-4,10(15)-dien-3-one (1) (1 g) was fed to M.
plumbeus in 20 flasks of each medium as outlined above.
After 10 days the fungus was harvested to give broth
extracts (0.532 and 0.613 g) and mycelial extracts (0.464
and 0.453 g) from HIM and LIM respectively. Analysis of
both extracts by TLC indicated the presence of bio-
transformed compounds. The broth and mycelial extracts
of each fermentation were combined and subjected to col-
umn chromatography using increasing concentrations
of EtOAc in petrol.
1
147.1175 (18), 133.1018 (55); H NMR: ꢀ 0.73 (3H, d,
J=7.6 Hz, H-13), 0.99 (3H, d, J=7.0 Hz, H-11), 3.50
(2H, dd, J=3.2, 7.3 Hz, H-14), 3.93 (1H, m, w=2=6.2
Hz, H-3), 4.50 (1H, bs, H-15), 4.64 (1H, d, J=1.6 Hz,
H-15).
3.4.7. (4S)-3ꢃ,12-Dihydroxycadin-10(15)-ene (8)
(HIM, 19.5 mg)
3.4.1. 14-Hydroxycadina-4,10(15)-dien-3-one (3)
(HIM, 34.4 mg)
Oil; ½ꢁŠ2D5:ꢁ0.91ꢂ (c=1.1, CHCl3); IR ꢂmax cmꢁ1 3426,
3304, 2929, 1716 cmꢁ1; EIMS m/z (rel. int.) 238.1928 (1)
[M]+, 220.1830 (31), 180.1515 (11), 162.1411 (70),
159.1175 (37), 149.0239 (100), 147.1175 (58), 133.1019
Oil; ½ꢁŠ2D5:ꢁ30.8ꢂ (c=1.6, CHCl3); IR ꢂmax cmꢁ1 3429,
2924, 1659; EIMS m/z (rel. int.) 234.1622 (3) [M]+,
216.1514 (19), 175.1121 (100), 174.1038 (26), 173.0965
(18), 149.0242 (16), 147.1175 (18), 133.1018 (16); 1H
NMR: ꢀ 0.81 (3H, d, J=7.0 Hz, H-13), 1.80 (3H, q,
J=1.6, Hz, H-11), 3.58 (2H, d, J=7.3 Hz, H-14), 4.54
(1H, bs, H-15), 4.75 (1H, m, w=2=4.4 Hz, H-15), 6.85
(1H, s, H-5);
1
(41); H NMR: ꢀ 1.03 (3H, d, J=6.3 Hz, H-11), 1.16
(3H, s, H-13), 1.20 (3H, s, H-14), 3.22 (1H, m,
w=2=15.0 Hz, H-3), 4.58 (1H, bs, H-15), 4.66 (1H, dd,
J=1.6, 3.5 Hz, H-15).
3.4.8. 13-Hydroxycadina-4,10(15)-dien-3-one (9)
(LIM, 62.4 mg)
3.4.2. (4S)-12-Hydroxycadin-10(15)-en-3-one (3)
(HIM, 58.7 mg)
Needles from acetone, mp 61–62 ꢂC; ½ꢁŠD25:ꢁ96.3ꢂ
(c=23.6, CHCl3) which was identified by comparison of
its physical and spectral data with those in the literature
(Buchanan and Reese, 2000).
Oil; ½ꢁŠ2D5:ꢁ76.8ꢂ (c=62.4, CHCl3); IR ꢂmax cmꢁ1
3320, 1728, 1679, 1451; EIMS m/z (rel. int.) 234.1620
(3.4) [M]+, 218.1671 (86), 175.1123 (32) 147.1174 (100);
1H NMR: ꢀ 0.80 (3H, d, J=7.0 Hz, H-13), 1.81 (3H, s,
H-11), 3.58 (2H, d, J=7.3 Hz, H-14), 4.54 (1H, s, H-15),
4.75 (1H, s, H-15), 6.89 (1H, 2, H-5).
3.4.3. (4S)-14-Hydroxycadin-10(15)-en-3-one (4)
(HIM, 23.0 mg; LIM, 13.8 mg)
Oil; ½ꢁŠ2D5:ꢁ15.3ꢂ (c=5.1, CHCl3) which was identi-
fied by comparison of its physical and spectral data
with those in the literature (Buchanan and Reese,
2000).
3.4.9. (4S)-10ꢁ,15-Epoxy-12-hydroxycadinan-3-one
(10) (LIM, 21.0 mg)
Oil; ½ꢁŠ2D5:ꢁ2.3ꢂ (c=19, CHCl3); IR ꢂmax cmꢁ1 3462,
1713, 1375, 1186, 1136; EIMS m/z (rel. int.) 252.1726 (9)
[M]+, 194.1307(15), 163.1123 (6), 161.0966 (14),
147.1174 (15), 145.1017 (6), 120.0939 (7), 119.0861 (6),

486
D.O. Collins et al. / Phytochemistry 59 (2002) 479–488
1
111.0810 (21), 105.0704 (9), 91.0548 (20); H NMR: ꢀ
1.03 (3H, d, J=6.5 Hz, H-11), 1.22 (1H, m, w=2=13.0
Hz, H-5), 1.24 (1H, m, w=2=7.5 Hz, H-8), 1.25 (3H,
s, H-13), 1.30 (3H, s, H-14), 1.32 (1H, m, w=2=10.0
Hz, H-7), 1.42 (1H, dt, J=3.5, 12.9 Hz, H-9), 1.78
(1H, dd, J=2.1, 3.5 Hz, H-1), 1.84 (1H, dt, J=1.2,
13.1 Hz, H-2), 1.92 (1H, tq, J=1.8, 3.0 Hz, H-9), 2.02
(1H, d, J=3.6 Hz, H-8), 2.05 (1H, t, J=2.8 Hz, H-6),
2.37(1H, d, J=3.6 Hz, H-4), 2.38 (1H, dd, J=3.7, 13.1
Hz, H-2), 2.48 (1H, d, J=4.0 Hz, H-15), 2.80 (1H, dd,
J=2.0, 4.0 Hz, H-15), 2.98 (1H, ddd, J=3.5, 4.5, 14.0
Hz, H-3).
w=2=12 Hz, H-6), 1.77 (1H, m, w=2=8 Hz, H-4), 1.79
(1H, m, w=2=10 Hz, H-2), 1.84 (1H, m, w=2=18 Hz,
H-8), 1.98 (1H, m, w=2=18 Hz, H-9), 3.31 (1H, d,
J=11.0 Hz, H-15), 3.50 (1H, d, J=11.5 Hz, H-15), 3.96
(1H, dd, J=3.0, 5.5 Hz, H-12).
3.5. Incubation of aromadendr-1(10)-en-9-one (14)
Aromadendr-1(10)-en-9-one (14) (1 g) was fed to M.
plumbeus in 20 flasks of HIM as outlined above. After 10
days the fungus was harvested to give broth (0.405 g) and
mycelial (0.832 g) extracts. Analysis of both extracts by
TLC indicated the presence of biotransformed com-
pounds. The extracts were combined and subjected to
column chromatography using increasing concentrations
of EtOAc in petrol.
3.4.10. (4S)-10ꢁ,15-Epoxy-14-hydroxycadinan-3-one
(11) (LIM, 9.8 mg)
Oil; ½ꢁŠ2D5: +6.9ꢂ (c=4.9, CHCl3); IR ꢂmax cmꢁ1 3421,
1713, 1230, 1028; EIMS m/z (rel. int.) 252.1726 (100)
[M]+, 234.1620 (7), 221.1542 (17), 220.1463 (24),
205.1229 (15), 194.1307 (67), 193.1229 (41), 176.1201
3.5.1. 2ꢃ-Hydroxyaromadendr-1(10)-en-9-one (15)
(57.7 mg)
Cubes from acetone, mp 85–86 ꢂC; ½ꢁŠD25:ꢁ186ꢂ
(c=6.3, CHCl3) which was identified by comparison of
its physical and spectral data with those in the literature
(Collins et al., 2001).
1
(46); H NMR: ꢀ 0.83 (3H, d, J=7.0 Hz, H-13), 1.02
(3H, d, J=6.3 Hz, H-11), 2.49 (1H, d, J=3.8 Hz, H-15),
2.79 (1H, dd, J=2.0, 4.1 Hz, H-15), 3.56 (1H, d, J=7.3
Hz, H-14), 3.56 (1H, d, J=7.3 Hz, H-14).
3.4.11. (4S)-10ꢁ,15-Epoxy-3ꢁ,14-dihydroxycadinane
(12) (LIM, 31.8 mg)
3.5.2. 2ꢁ-Hydroxyaromadendr-1(10)-en-9-one (16)
(53.5 mg)
Cubes from acetone, mp 72–74 ꢂC; ½ꢁŠD25:ꢁ91ꢂ
(c=18.6, CHCl3) which was identified by comparison of
its physical and spectral data with those in the literature
(Collins et al., 2001).
Oil; ½ꢁŠ2D5: +45.7ꢂ (c=12.7, CHCl3); IR ꢂmax cmꢁ1
3404, 1029, 991; EIMS m/z (rel. int.) 254.1882 (20)
[M]+, 236.1776 (15), 205.1592 (10), 178.1358 (28),
1
149.0239 (100); H NMR: ꢀ 0.77 (3H, d, J=7.0 Hz, H-
13), 0.98 (3H, d, J=7.0 Hz, H-11), 0.98 (1H, m,
w=2=18.0 Hz, H-2), 1.09 (1H, t, J=12 Hz, H-5), 1.20
(1H, m, w=2=15.0 Hz, H-6), 1.25 (1H, m, w=2=15.0
Hz, H-8), 1.36 (1H, dt, J=4.0, 12.5 Hz, H-9), 1.44 (1H,
dt, J=3.5, 10.5 Hz, H-7), 1.49 (1H, m, w=2=20.0 Hz,
H-4), 1.63 (1H, m, w=2=11.5 Hz, H-5), 1.66 (1H, m,
w=2=20.0 Hz, H-8), 1.84 (1H, dt, J=3.0, 13.5 Hz, H-2),
1.94 (1H, tq, J=2.0, 12.5 Hz, H-9), 2.01 (1H, ddd,
J=3.3, 10.1, 10.3 Hz, H-1), 2.08 (1H, w=2=13.4 Hz, H-
12), 2.43 (1H, d, J=4.6 Hz, H-15), 2.76 (1H, dd, J=2.0,
2.6 Hz, H-15), 3.51 (1H, dd, J=3.2, 9.7Hz, H-14), 3.54
(1H, dd, J=3.2, 9.7Hz, H-14), 3.85 (1H, d, J=2.5 Hz,
H-3).
3.5.3. 13-Hydroxyaromadendr-1(10)-en-9-one (17)
(52.6 mg)
Oil; ½ꢁŠ2D5: ꢁ118ꢂ (c=4.4, CHCl3) which was identified
by comparison of its physical and spectral data with
those in the literature (Collins et al., 2001).
3.5.4. 2ꢃ,13-Dihydroxyaromadendr-1(10)-en-9-one (18)
(48.1 mg)
Needles from acetone, mp 145–146 ꢂC; ½ꢁŠD25:ꢁ128ꢂ
(c=0.6, CHCl3) which was identified by comparison of
its physical and spectral data with those in the literature
(Collins et al., 2001).
3.4.12. (4S)-3ꢁ,10ꢁ,12,15-Tetrahydroxycadinane (13)
(LIM, 23.2 mg)
3.5.5. 2ꢁ,13-Dihydroxyaromadendr-1(10)-en-9-one (19)
(33.5 mg)
Oil; ½ꢁŠ2D5: +70.2ꢂ (c=9.9, CHCl3); IR ꢂmax cmꢁ1
3420, 1379; EIMS m/z (rel. int.) 254.18820 (30) [M–
H2O]+, 236.1776 (4), 223.1698 (6), 168.1514 (10),
Oil; ½ꢁŠ2D5: ꢁ101.0ꢂ (c=0.5, CHCl3); IR: ꢂmax cmꢁ1
3457, 2928, 1632; UV (CHCl3) lmax nm (log ") 231
(2.69); EIMS m/z (rel. int.) 250.1569 (22) [M]+,
204.1511 (29), 162.1042 (40), 149.0964 (41), 119.0860
(45), 107.0861 (100); 1H NMR: ꢀ 0.99 (1H, m, w=2=9.2
Hz, H-6), 1.13 (3H, d, J=7.0 Hz, H-15), 1.29 (3H, s, H-
12), 1.65 (1H, dd, J=3.3, 7.0 Hz, H-3), 1.96 (3H, bs, H-
14), 2.22 (1H, m, w=2=15.8 Hz, H-8), 2.89 (1H, dd,
J=5.1, 15.2 Hz, H-8), 3.39 (2H, d, J=3.2 Hz, H-13),
4.82 (1H, t, J=13.3 Hz, H-2).
1
153.0916 (11), 142.0994 (43), 111.0810 (100); H NMR:
ꢀ 1.02 (3H, d, J=7.0 Hz, H-11), 1.25 (3H, s, H-13), 1.27
(1H, d, J=4.0 Hz, H-7), 1.31 (3H, s, H-14), 1.35 (1H,
dq, J=2.0, 7.0 Hz, H-5), 1.37 (1H, ddd, J=0.5, 10.5,
13.0 Hz, H-9), 1.49 (1H, dd, J=12.0, 23.5 Hz, H-5), 1.54
(1H, dd, J=2.5, 12.0 Hz, H-2), 1.61 (1H, tt, J=1.5, 12.5
Hz, H-1), 1.69 (1H, m, w=2=18 Hz, H-8), 1.72 (1H, m,

D.O. Collins et al. / Phytochemistry 59 (2002) 479–488
487
3.6. Incubation of methyl 3ꢃ-hydroxyurs-12-en-28-oate (21)
Aranda, G., El Kortbi, M.S., Lallemand, J., Neuman, A., Ham-
moumi, A., Facon, I., Azerad, R., 1991a. Microbial transformation
of diterpenes: Hydroxylation of sclareol, manool and derivatives by
Mucor plumbeus. Tetrahedron 47, 8339–8350.
Methyl 3b-hydroxyurs-12-en-28-oate (21) (500 mg)
was fed to M. plumbeus in 20 flasks of HIM as outlined
above. After 10 days the fungus was harvested to give
broth (0.155 g) and mycelial (0.550 g) extracts. Analysis
of both extracts by TLC indicated the presence of bio-
transformed compounds. The extracts were separately
subjected to column chromatography using increasing
concentrations of EtOAc in petrol.
Aranda, G., Lallemand, J., Hammoumi, A., Azerad, R., 1991b.
Microbial hydroxylation of sclareol by Mucor plumbeus. Tetra-
hedron Letters 32, 1783–1786.
Arantes, S.F., Farooq, A., Hanson, J.R., 1999a. The preparation and
microbiological hydroxylation of the sesquiterpenoid nootkatone.
Journal of Chemical Research, (S) 248.
Arantes, S.F., Hanson, J.R., 1999. The hydroxylation of the sesqui-
terpenoid guaioxide by Mucor plumbeus. Phytochemistry 51, 757–
760.
The mycelial extract contained, almost exclusively,
untransformed methyl ursolate (21). When chromato-
graphed, the broth was found to contain methyl
3b,7b,21b-trihydroxyursa-9(11),12-dien-28-oate (22) (6.4
mg): Needles from acetone, mp 123–125 ꢂC, ½ꢁŠD25:
+164.2ꢂ (c=6.4, CHCl3); IR: ꢂmax 3406, 1715, 1456,
1199 cmꢁ1; EIMS m/z 500.3502 (52) [M]+, 482.3396
Arantes, S.F., Hanson, J.R., Hitchcock, P.B., 1999b. The preparation
and microbiological hydroxylation of 10b,11-oxido-4a,5a,7b-ere-
mophilane. Journal of Chemical Research, (S) 314–315.
Arantes, S.F., Hanson, J.R., Hitchcock, P.B., 1999c. The hydroxyla-
tion of the sesquiterpenoid valerianol by Mucor plumbeus. Phy-
tochemistry 52, 1063–1067.
Arantes, S.F., Hanson, J.R., Hitchcock, P.B., 1999d. The micro-
biological hydroxylation of the sesquiterpenoid patchoulol by
Mucor plumbeus. Phytochemistry 52, 635–638.
1
(100), 468.3240 (15), 440.3290 (25), 427.2848 (88); H
NMR: ꢀ 0.81 (3H, s, H-24), 0.86 (1H, m, w=2=11.5 Hz,
H-5), 0.92 (3H, d, J=6.5 Hz, H-29), 0.96 (3H, s, H-27),
0.98 (1H, d, J=2 Hz, H-20), 1.00 (3H, s, H-26), 1.03
(3H, s, H-23), 1.08 (3H, d, J=6.3 Hz, H-30), 1.18 (3H,
s, H-25), 1.30, m, w=2=13.3 Hz, H-15), 1.32 (1H, m,
w=2=12.5 Hz, H-1), 1.43 (1H, m, w=2=12 Hz, H-19),
1.50 (1H, dd, J=3.8, 14.9 Hz, H-22), 1.52 (1H, dd,
J=11.3 14.1 Hz, H-6), 1.64 (1H, m, w=2=30 Hz, H-2),
1.73 (1H, m, w=2=20 Hz, H-2), 1.80 (1H, dd, J=5, 12.5
Hz, H-6), 1.87(1H, dd, J=4.3, 9.3 Hz, H-16), 1.91 (1H,
m, w=2=7.5 Hz, H-16), 1.98 (1H, dt, J=4.8, 10.9 Hz,
H-1), 2.09 (1H, dd, J=4.5, 12.5 Hz, H-22), 2.17(1H, dt,
J=5.8, 13.8 Hz, H-15), 2.41 (1H, d, J=10.1 Hz, H-18),
3.23 (1H, dd, J=4.6, 11.7Hz, H-2), 3.45 (1H, m,
w=2=25 Hz, H-21), 3.66 (3H, bs, CO2Me), 4.05 (1H, dd,
J=5.4, 11.4 Hz, H-7), 5.58 (1H, d, J=6.0 Hz, H-12),
5.63 (1H, d, J=6.0 Hz, H-11).
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Acknowledgements
This work was supported in part by funds secured
under the University of the West Indies/Interamerican
Development Bank (UWI/IDB) Programme and the
granting of a UWI Postgraduate Scholarship to DOC
and a Research Fellowship to PLDR. The authors are
grateful to Professor John C. Vederas (University of
Alberta) for mass spectral analyses and Professor Her-
bert L. Holland (Brock University) for helpful discus-
sions. Optical rotations were measured at the Bureau of
Standards, Kingston. Fermentations were carried out in
the Biotechnology Centre, UWI.
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